Method and device for identification of nucleated red blood cells from a maternal blood sample

ABSTRACT

A method for concentrating and isolating nucleated cells, such as a maternal and fetal nucleated red blood cells (NRBC&#39;s), in a maternal whole blood sample. The invention also provides methods and apparatus for preparing to analyze and analyzing the sample for identification of fetal genetic material as part of prenatal genetic testing. The invention also pertains to methods and apparatus for discriminating fetal nucleated red blood cells from maternal nucleated red blood cells obtained from a blood sample taken from a pregnant woman.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of and claims priority ofU.S. patent application Ser. No. 13/046,543, filed on Mar. 11, 2011,which claims the benefit under 35 U.S.C. §119(e) of U.S. ProvisionalAppl. No. 61/313,098, filed Mar. 11, 2010, the disclosures of which areincorporated herein by reference.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BACKGROUND OF THE INVENTION

Prenatal genetic testing requires access to fetal DNA. As described incommonly owned US Patent Appl. Publ. No. 2010/0159506, fetal geneticmaterial can be found within fetal cells present in the mother'scirculating blood. These fetal cells originate in the fetus and crossthe placenta to enter the mother's circulatory system.

Maternal blood contains both nucleated (i.e., containing geneticmaterial) and non-nucleated cells of both fetal and maternal origin. Inorder to focus attention on the cells of most interest, a first step infetal genetic testing may therefore be to concentrate nucleated cellswithin the sample. One prior approach is described in US Patent Appl.Publ. No. 2010/0159506. Since red blood cells are denser than whiteblood cells, a preliminary separation of red blood cells is obtained bya single density gradient to separate mononuclear cells, includingnucleated red blood cells, from a whole blood sample. The sample is thenapplied to a slide in a monolayer, stained, and analyzed.

SUMMARY OF THE INVENTION

The present invention provides a method for concentrating and isolatingnucleated cells, such as maternal and fetal nucleated red blood cells(NRBC's), from a maternal whole blood sample. The invention alsoprovides methods and apparatus for preparing to analyze and analyzingthe sample for identification of fetal genetic material as part ofprenatal genetic testing. The invention also pertains to methods andapparatus for discriminating fetal nucleated red blood cells frommaternal nucleated red blood cells obtained from a blood sample takenfrom a pregnant woman.

One aspect of the invention provides a method of enriching for fetalnucleated cells (such as, e.g. nucleated red blood cells) from amaternal blood sample. In some embodiments, the method includes thefollowing steps: passing a maternal blood sample containing maternalnucleated cells and fetal nucleated cells through a filter (such as,e.g., a leukocyte depletion filter); retaining nucleated cells on thefilter; and eluting nucleated cells from the filter with an elutionbuffer wherein the nucleated cells include fetal nucleated cells.

In some embodiments, the method also includes the step of staining theblood sample, such as by staining the blood sample with a nuclear stain.

In some embodiments, the method also includes the step of concentratingand resuspending the blood sample before the passing step, such as bycentrifugation.

In some embodiments, the method also includes the step of measuring theblood sample before concentrating to, e.g., standardize an amount ofcells in the blood sample.

Another aspect of the invention provides a method of creating a layer ofcells on a surface including the following steps: moving a sample ofcells in at least two directions relative to a surface to create amonolayer of cells on the surface; and adhering the cells to thesurface. The movement may include, e.g., circular movement, zigzagmovement, diagonal movement and/or serpentine movement. The relativemovement may also include moving a portion of the sample away from thesurface.

Some embodiments employ a smear tool in the moving step. In some suchembodiments, the angle of the tool with respect to the surface may bevaried. The relative speed of the tool with respect to the surface mayalso be varied, for example, in the range of 0.1 mm/sec to 500 mm/sec.

The moving step may include the step of generating a generally uniformsample density. Some embodiments also include the step of monitoring thedensity of the sample relative to the surface, such as by using red orblue light.

Yet another aspect of the invention provides a method of identifying anucleated fetal cell. In some embodiments the method includes the stepsof: adhering nucleated cells from a maternal blood sample to a surface,the surface comprising a plurality of portions; generating a pair ofimages corresponding to at least one portion of the surface; applying analgorithm to the pair and determining if the portion includes a cell ofinterest; and performing an analysis using a fetal identifier on atleast one portion of the surface that includes a cell of interest andthereby determine if the at least one portion contains a fetal cell. Insome embodiments, the analysis is selected from the group consisting ofin situ hybridization and immunohistochemistry, and the method furtherincludes the step of scanning the surface with an automated microscopeafter the adhering step. Some embodiments include the step of generatinga third image.

In some embodiments, the step of generating a pair of images furtherincludes the step of generating a first image with transmittedillumination and a second image with coaxial illumination. In variousembodiments the wavelength of the transmitted illumination may bebetween 380 nm and 800 nm, above 620 nm, or around 420 nm. The coaxialillumination may be between 350 nm and 364 nm.

In some embodiments, the step of performing an analysis includes thestep of selectively placing fetal identifiers on a plurality of portionsof the surface, wherein each portion contains a candidate fetal cell.

In some embodiments, the step of applying an algorithm includes thesteps of flattening at least one image; segmenting at least one imageand thereby define foreground and background pixels; removing backgroundpixels from at least one image to generate a transformed image;enumerating nuclei in the transformed image to generate enumeratednuclei; and calculating at least one of complexity and brightness for atleast one enumerated nuclei, wherein low complexity or high brightnessindicate a fetal cell character.

In some embodiments, the applying step further includes the steps ofcalculating a brightness of a background of the surface and a brightnessof a foreground of a surface, and comparing a measurement of the pair ofimages to one or both of the background brightness and the foregroundbrightness.

Some embodiments include the additional step of storing a location ofthe pair of images. A nucleated cell may be located based on such astored location.

Some embodiments include the step of fixing the sample with a non-crosslinking fixative before the performing an analysis step and/or fixingthe sample at a reduced temperature before the performing an analysisstep.

In some embodiments, the performing step may include the step oftreating with a stabilizer and/or treating with an antibody selectedfrom the group consisting of anti zeta hemoglobin and anti epsilonhemoglobin if the analysis is immunohistochemistry.

Still another aspect of the invention provides a method of identifying agenetic status of a fetus. In some embodiments the method includes thefollowing steps: adhering nucleated cells from a maternal blood sampleto a surface, the surface comprising a plurality of portions; generatinga pair of images corresponding to at least one portion; applying analgorithm to the pair of images and determining if the portion includesa cell of interest; performing an analysis using a fetal identifier onat least one portion of the surface that includes a cell of interest andthereby determine if the at least one portion contains a fetal cell(such as, e.g., by in situ hybridization and/or immunohistochemistry);and performing an analysis using a genetic identifier on at least oneportion of the surface that includes a fetal cell and thereby determinethe genetic status of the fetus (such as, e.g., by RNA in situhybridization, DNA in situ hybridization and/or immunohistochemistry).The two performing steps may be performed at the same time.

Some embodiments may include the additional step of applying a pressureon the surface that is lower than atmospheric pressure during theperforming step(s). The method may also include the step of crushing atleast one cell of interest and an associated nuclei prior to theperforming the analysis step.

Yet another aspect of the invention provides a method of identifying afetal cell. In some embodiments, the method includes the followingsteps: providing a maternal blood sample; and performing in situhybridization using a TSIX probe on the sample to generate a signalwherein a positive signal using a TSIX probe is indicative of thepresence of fetal cellular material. The method may also include thestep of separating the sample to generate a plurality of portions beforethe performing step to, e.g., provide a monolayer of cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe claims that follow. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIGS. 1-2 show a process and device for enriching for fetal nucleatedcells from a blood sample using a filter system.

FIG. 3 shows a cell smear tool creating a layer of cells on a surface.

FIG. 4 shows a system for automating the process using a device such asthe one shown in FIG. 1.

FIG. 5 shows an automated system used to identify fetal nucleated cells.

FIG. 6 shows cell analysis results after enriching for fetal nucleatedcells according to one aspect of the disclosure.

DETAILED DESCRIPTION OF THE INVENTION

Leukocyte depletion filters can be used to filter blood products fromwhole blood. Information about leukocyte depletion filters may be foundin the following: U.S. Pat. Nos. 5,676,849; 5,662,813; 6,544,751;4,923,620; 4,925,572; Comparison of Five Different Filters for Removalof Leukocytes from Red Cell Concentrates; VOX Sang 1992/62:76-81;Recovery of Human Leukocytes from a Leukodepletion Filter; Chang et al;J. Transfusion 1992/32 (85); Recovery of Functional Human Lymphocytesfrom Leukotrap Filters; Longley et al.; J. Immunological Methods;1999/121:33-38; Biotechniques 31:464-466 (2001); S. Ebner et al.; J.Immunological Methods; 2001(252):93-104. In one aspect of the invention,such filters can be used to separate nucleated blood cells fromnon-nucleated blood cells.

According to one embodiment, a first step of the method is filtration.The incoming blood sample is filtered (e.g., within 24 hours of draw,preferably within 12 hours of draw) using a leukocyte depletion filter.A suitable leukocyte depletion filter is the PureCell™ Select Systemfrom Pall Corporation.

Custom chemistry may be used for cell suspension during the filtrationprocess. The chemistry may include trehalose, maltose, dextran,pseudoephedrine, fluoride, phosphate and/or sulfate. The chemistry mayaddress issues related to the degradation of cells during processingusing the manufacturer's recommended process; improve the cellmorphology in the final sample and may enable the unique DAPI+420 nmimaging process; reduce cell clumping and sphering which improves theability to spread the cells evenly in a large area monolayer; stabilizethe cells and provide more margin in terms of the time between the startof the filtration process and the creation of the monolayer; and reducecell destruction and release of DNA into the suspension. The chemistrymay also be used to increase the allowable time between blood draw andprocessing which will expand the population of candidate patients byopening the test to doctors in areas that are not near metropolitancenters.

FIGS. 1-2 show a process and device for enriching for fetal nucleatedcells from a blood sample using a filter system according to oneembodiment of the invention. A maternal blood sample 6 containingvarious components of red blood including nucleated maternal and fetalnucleated cells 7 such as fetal nucleated red blood cells, is containedin bag 4. Valve 28 connecting tubing 8 to collection bag 30 and valve 24connecting tubing 20 to tubing for elution 26 are closed. Valve 10connecting tubing 8 to filter 12 is opened and blood sample 6 is passedthrough filter 12. Cells for collection 14, including nucleated fetalcells 7, remain behind on filter 12, while unwanted blood products 18,including mature reticulocytes, pass through filter 12 and are collectedin waste collection bag 16.

After all of the unwanted blood products 18 (e.g., non-nucleated cells)have passed through the filter, the nucleated cell population remainingon the filter contains the cells of interest, NRBC's, as asubpopulation. As shown in FIG. 2, valves 10 and 22 are closed. Asyringe 36 containing elution solution 40 is attached to tubing 26.Valves 24 and 28 are opened. A syringe 36 containing elution solution 40is attached to tubing 26. Valves 24 and 28 are opened. Elution fluid 40is forced backward through the filter and causes the nucleated cells 7to be released from the filter. An elution fluid such as one recommendedby the manufacturer of the filter may be used. Alternatively, one aspectof the invention includes use of an elution fluid containing one or moreof sodium or potassium fluoride (e.g., 0.01 to 100 mg/mL),pseudoephedrine (e.g., 0.1 to 100 mg/L), EDTA (e.g., 1 to 10 mM), ACD-A(e.g., 0.01 to 10%) trehalose (e.g., 0.01 to 10 gm/L), maltose (e.g.,0.01 to 10 gm/L), dextran (e.g., 0.01 to 3.0%, 100-500 MW), F-68 (e.g.,0.001 to 10 mg/mL), sodium or potassium sulfate (e.g., 1 to 100 mM), orsodium or potassium phosphate (e.g., 1 to 100 mM). In one embodiment,the elution solution includes about 25 ug maltose and about 75 ugtrehalose in about 25 mL phosphate buffered solution (PBS). Elutionfluid 40 and cells 7 are collected in collection bag 30 to yield anenriched fetal nucleated cell population 42. These released cells andthe elution fluid are collected into vials.

Machinery may be provided to automate the filtration and cell releaseprocess. The machinery may control either or both of the flow rate andamount of fluid that moves through the filter system. FIG. 3 showscontroller 70 according to an embodiment of the invention configured tocontrol flow rates of sample, waste, elution fluid, and/or elution fluidcontaining fetal nucleated cells through valves 10, 28, 22, and/or 24. Aflow rate equal to 0.2-50 ml/second may be used. The volume of fluidthat moves through the filter system may be from 5 mL to 500 mL.Controlling flow rates can have a significant impact on the cellpopulation that is recovered: If the flow rate is too high, cells ofinterest may be driven deeply into the filter making them harder torecover. Also, if the flow rate is too high, cells may be damaged byhydrodynamic forces. If the flow rate is too low on the releasebackflush, cells of interest may not be recovered; they will remaintrapped by the filter. Machinery for automation of filtration and cellrelease process may include a computer controlled valve and pump systemwhereby the fluid flow paths through the filtration device can becontrolled by a series of valves and manifolds. One type of valveparticularly well suited is the pinch tube valve since it can controlfluid flow without coming in contact with the fluids. This isadvantageous for avoiding cross-contamination between samples and makingfor a fully disposable filtration system where expensive components suchas valves do not need to be discarded or cleaned. The flow through thefiltration device can be accomplished by a combination of gravity andmechanical pump systems such that both rate of flow and flow velocityprofiles can be set and controlled by computer hardware and software.

The machinery may also improve the repeatability of the process andreduce cycle time. For example, the automation may enable high speedprocessing while maintaining standardization of sample handling. Also,the filtration process requires control of valves, volumes, and flowrates during the process. The automation will minimizeoperator/technique dependent variability.

The next step may be concentration by centrifugation. The cells may bestained and counted (e.g., using the optical density of the suspensionas a measure of cell count) before centrifugation. Alternatively, oradditionally, cells may be stained after centrifugation. After the cellshave been collected in a centrifuge tube (having, e.g., total volume ofapproximately 50 ml dilute cell suspension), the tube is spun in acentrifuge (e.g., for 15 minutes at 500×g at room temperature) toconcentrate the cells into a pellet at the bottom of the tube. Theresulting pellet volume is approximately 100 μl to 300 μl. It ispossible to stain the cells before centrifugation as well as count thenumber of cells in the pre-centrifuge suspension.

Measuring the cell count in the pre-centrifuge suspension using theoptical density of the suspension allows the automation of the removalof supernatant and the automation of the addition of stabilizers andstain (e.g., 0.01-10% ACD-A, 0.01 to 3% of 10 K-400 K MW dextran, 1-10mM EDTA, 0.01 to 10 g/L maltose, 0.1 to 100 mg/mL pseudoephedrine, 0.01to 10 g/L Trehalose, 0.01 to 100 mg/mL sodium or potassium fluoride, 1to 100 mM sodium or potassium phosphate, and/or 1 to 100 mM sodium orpotassium sulfate. In one embodiment, stabilization fluid may containsodium sulfate (less than about 20 grams per L), sodium chloride (lessthan about 2 grams per L), imidazole (less than about 3 grams per L),sodium fluoride (about 2 grams per L), and pseudoephedrine (less thanabout 0.1 grams per L). In some cases, measuring the cell count aftercentrifugation may lead to large errors in cell counts because thevolume is so small and the optical density is very high (opaque).

Automating the volume of stain and stabilizer that is added to thesuspension prior to centrifugation allows the stain and stabilizer toreach and affect all cells in a more homogeneous manner. Adding stainand stabilizer after centrifugation in some cases may lead tonon-homogeneous staining and stabilization because the cells havealready started to clump and the large solid to liquid ratio in thepellet may inhibit the even distribution of the additives.

Adding a nuclear stain prior to the creation of the monolayer reducesthe number of processing steps required to enable scanning In somecases, staining after the monolayer has been created requires a fixationstep that adds complexity, handling, and cost to the sample processingand may modify the cell morphology, reducing the effectiveness of theautomated digital microscopy.

Automating the amount of supernatant that is removed aftercentrifugation may improve the repeatability of the final cellsuspension that is used to create the monolayer. Standardizing the solidto liquid ratio of the monolayer suspension generates more uniformdistribution of cells on each substrate and also improves thevariability between individual smears on slides. Maximizing the celldistribution uniformity enables the extrapolation of one slide's resultsto all slides in a single patient set. This ability to accuratelyextrapolate results means that a smaller number of slides must be putthrough the imaging system and reduces the necessary cycle time perpatient. In addition, tight control of the suspension density minimizesor eliminates the need for a trial and error approach to creating amonolayer with an appropriate cell density for automated digitalmicroscopy.

After concentration in the centrifuge, the cells are resuspended. Usingthe cell count as a guide for the final required liquid volume, allunwanted suspension fluid is removed from the centrifuge vial until onlythe desired total volume remains in the vial. The remaining supernatantand cells are then gently mixed (e.g., by agitation or by repeatedaspiration and dispense cycles using a pipette). Automated removal ofthe supernatant based on the pre-centrifugation cell count enables thecreation of a monolayer that is optimized for automated digitalmicroscopy and rare cell identification. Next, a monolayer is created.The resuspended pellet is then dispensed onto a substrate, such as clearglass of standard microscope slide thickness. Using an automated smeartool, this droplet is spread evenly across the substrate surface in amanner that minimizes clustering and overlap of cells while maximizingthe number of cells per unit area. FIG. 4 shows a cell smear tool 50according to one embodiment of the invention. A sample of enriched fetalnucleated cells 52 is placed on surface 54. Cell smear blade 56 is movedby cell smear tool controller 60 across sample of enriched fetalnucleated cells 52 to create a layer of cells 66 on surface 54.

Prior automated smearing tools merely recreated the human motion thathas historically been used to create blood smears by moving only with asingle action linear speed motion in one axis (e.g., along the long Yaxis of the slide). In one aspect of the invention, the automated smeartool moves in both X and Y while making essentially a monolayer smear.The motion patterns of a stage (e.g., an XY motorized stage) controlledby the computer software are unique. In some embodiments, the motiongoes across the short X axis of the slide. The use of the short X axismotion can be helpful in creating a suspension along the smear slidebefore actual forward motion of the smear begins. In some embodiments,the software runs open loop (without monitoring cell density).

In other embodiments, cell density is monitored. In FIG. 4 light 62illuminates layer of cells 66 and images are captured by detector 64 andused to determine cell density. In one embodiment, feedback fromdetector 64 is used to guide controller 60 to change parameters of cellsmear blade 56 to control the density of cells 66 on surface 54. Thesmear density could be monitored, e.g., by using blue light (which willbe absorbed by the hemoglobin) or red light (which will measure allcells). Higher attenuation of the light indicates a denser cell layer;lower attenuation indicates a less dense cell layer. In someembodiments, the system is configured to vary parameters such as angleof smear and/or speed of smear in real time to assure that theuniformity of the monolayer is maintained.

The automated smear tool varies its speed during the monolayer smearingprocess to control the density of cells that are applied per unit areato the substrate. The software speed may vary, for example, from 0.1mm/sec to 500 mm/sec. The software may have a starting speed and endingspeed for the smear tool which are the same, or they may differ fromeach other. In the case where the starting speed and ending speedsdiffer, the smear tool may change speed automatically in real timeduring the smear process. The rate of change may be linear orexponential. It could also vary sinusoidally or be varied using anyother continuous function during the smear. The automated smear tooloptimizes the initial droplet pickup and spread more or less normal tothe main direction of smear. This may improve the overall uniformity ofthe smear normal to the main direction of smear.

The automated smear tool may move in a zigzag pattern during thesmearing process to improve the homogeneity of the suspended cellpopulation and the homogeneity of the cell population distributions perunit area. The automated smear tool can create smears in any size, suchas 1″×2″ or 5″×5″ (125 mm×125 mm).

The smear tool may have any motion that aids in distributing the cells.The smear tool may move in both the X and Y directions during both thepickup of the initial cell droplet and during the actual smearingprocess itself. The motion(s) of the smear tool may be circular, zigzag,forward, backward, side to side with no forward or backward motion,diagonal, serpentine (move in +X, move in +Y, move in −X, move in +Y,repeat). These motions are useful for two reasons: they make the heightof the meniscus even across the face of the smearing tool and improvethe uniformity of the cell density in the direction normal to the mainaxis of the smear. Second, the starting and stopping motion helps tomove cells up into suspension and improve the homogeneity of the cellpopulations distribution within the meniscus behind the smearing tool.

The automated smear tool may be controlled by a GUI that allows the userto set parameters such as smear speed, velocity profile, cross motion Yaxis suspension parameters and motion distance. The parameters may beset up and stored based on an initial test smear.

The smear tool may have notched edges or polished edges, or it may beuncharged or have varying surface charge. The smear tools and/or smearslides may be made of glass or material other than glass.

The monolayer is then imaged using an automated microscopy platform. Ingeneral any microscopy platform may be used with the requirement thatthe microscopy platform has the capability to provide transmittedillumination (e.g., 380 nm-800 nm range) and coaxial illumination (e.g.,in the 350 nm-364 nm range to excite the nuclear stain). FIG. 5 shows anautomated microscope system to identify fetal nucleated cells accordingto one aspect of the invention. Light 86 or light 94 is delivered tosurface 82 containing fetal nucleated cells 84 and other cells. Lightfrom the cells on surface 82 is detected through microscope 90.Microscope 90 is moved across surface 82 by controller 92.

A pair of images is acquired for each XY location on the substrate. Forexample, one image uses 420 nm illumination, and one image uses thefluorescent illumination. The XY locations are selected in such a waythat the entire area of the substrate is imaged. For each location, theimage pair is used to determine locations where the 420 nm light isabsorbed which indicates the presence of hemoglobin. The fluorescentimage is used to determine the location of nuclear material in the fieldof view. Where the characteristics of shape and brightness of thenucleus match the known characteristics of an NRBC and hemoglobin isalso present, an NRBC candidate has been found and this candidate isadded to a list of candidate cells.

Note that red light in the range of 620 nm or higher may also be usedsince this red light will not be absorbed by either nuclear material orby hemoglobin. The red image will thus only contain the actual physicalstructure of the cells: cell edges, texture or background artifacts fromthe suspension medium, and non-cell particles on the slide. This truestructure information may be used when determining the amount ofhemoglobin signal that is coincident with nucleus locations in order toreduce false positives. (It is not possible to separate signal generatedby structural edges from signal generated due to 420 nm absorbance usingthe 420 nm/fluorescent image pair only.) The initial imaging may be doneat a low magnification (e.g., 10×) to improve system speed and thehighest scoring. NRBC candidates are revisited at higher magnificationto verify the status as true NRBCs.

A high speed automated digital microscopy platform may be used tocollect the images. Any set of image pairs (e.g., 420 nm/fluorescent) ortriads (e.g., 420 nm, 630 nm, fluorescent) can be used to sort the cellsinto subpopulations of white cells, non-nucleated red cells, andnucleated red cells. The cell identification and subpopulation groupingalgorithm does not interrogate cells that are known to be outside thepopulation of interest.

The combination of the 420 nm and fluorescent imaging is made possibledue to the sample preparation steps that preserve the morphology of thecells and maintain the hemoglobin intact through the creation of themonolayer. A unique algorithm can be used to determine the NRBCcharacter of any cell. The heart of the algorithm is the ability todetermine the coincidence of 420 nm absorption and nuclear fluorescence(completely dark NRBCs) in addition to the adjacency of 420 nmabsorption to nuclear fluorescence. The steps of the algorithm mayinclude:

-   -   Flatten the illumination (make the image brightness even across        the complete image to correct for dimness in the image corners        which may arise due to optical system aberration).    -   Apply the image flattening to both fluorescent and 420 nm        transmission images.    -   Segment the images to determine which pixels belong to        foreground (nuclei and hemoglobin) and which pixels belong in        the background. Image segmentation removes noise from the images        and may reduce the number of pixels that must be processed in        the remaining steps.    -   Enumerate (count and number sequentially) the individual nuclei        in the fluorescent image.    -   Calculate their complexity and average brightness (#edge        pixels²)/(# interior pixels) tends to 4π or 12.56 for perfectly        round nuclei. NRBC nuclei tend to be round (low complexity) and        bright.    -   For the subpopulation of the nuclei with the lowest complexity,        determine the hemoglobin absorbance coincident to the nuclei and        the hemoglobin absorbance around the perimeter of the nuclei.        For example, the 50,% 25%, or 10% least complex are examined.        (The higher nuclei do not need to be examined.)

Any parameters of the nuclei may be examined. In some embodiments, thebrightness, complexity, and/or hemoglobin absorbance of the nuclei aremeasured. The cells that cluster in the 3-space region of highhemoglobin absorbance, low complexity, and high brightness have a highlikelihood of being NRBCs and are graded as very likely candidates to beNRBCs.

In a similar manner the white cells may be binned into theirsubpopulations (e.g., segmented neutrophils have high complexity andlarge whites), and every WBC that is put into a specific bin removesanother cell from the NRBC candidate pool.

Both hemoglobin absorbance and nuclear fluorescence signals may becalculated relative to the average brightness of the foreground and theaverage brightness of the background. In this manner it is possible tomake comparisons intra-site. (One pair of 420 nm/fluorescent images canbe compared to another pair of 420 nm/fluorescent images.) If relativemeasures are not used then variations larger than a single field of viewwill impact the ability to correctly detect the cells of interest acrossthe entire slide. Examples of large scale variations include: variationsin fluorescent staining, variations in RBC hemoglobin lysing, andillumination changes (lamp warming up or failing).

The storage of the NRBC candidate XY locations enables the use of lessFISH reagent which reduces cost. The FISH reagents may be applied onlyto the sites where NRBCs have been located. After FISH is complete,these XY locations are used again to revisit the NRBCs and interrogatethe final genetic testing result.

The XY locations of the candidate NRBCs may also be used to control amicrodissection system. Microdissection may be used to pick up the NRBCsand physically segregate them away from the non-NRBC cell population.This microdissection enrichment process may be used to provide highpurity DNA samples for use in microarray applications, PCR, or other DNAanalysis methods.

The image can then be analyzed. The use of the 420 nm and fluorescentimage pairs (or a 420 nm, 630 nm, and fluorescent image trio) to findNRBC candidates depends on the following characteristics of NRBCs:

-   -   (1) NRBCs are associated with hemoglobin absorbance (dark pixels        in the 420 nm image). Sometimes the hemoglobin absorbance area        covers the same set of pixels as the nuclear fluorescence. In        other cases the hemoglobin absorbance is adjacent to the        fluorescent signature of the nuclear material.    -   (2) NRBC nuclei tend to be single nuclei and tend to be round        (low complexity)    -   (3) NRBC nuclei tend to have a brighter fluorescent signal that        other cell nuclei in the same image    -   (4) NRBC nuclei tend to be smaller than most other nuclei        because they are in the process of condensing and being forced        out of the cell (e.g., by apoptosis)

Next is the fixation step. The cells are treated with a non-crosslinkingfixation in preparation for FISH. This fixation is performed in thepresence of stabilizers which improve the FISH results, maintain RBCmorphology and hemoglobin signal for later relocation/revisit of thefetal NRBCs using the previously stored XY locations. Fixation mayinclude EtOH (40 to 90%), glyoxal (0.1 to 25%), methanol (0.1 to 10%)and or isopropanol (0.1 to 10%) for (15 sec to 10 min) preceded by a −20degree centigrade methanol dip for 30 seconds to 2 days.

Stabilizers may include Sodium or potassium fluoride (0.01 to 100mg/mL), pseudoephedrine (0.1 TO 100.0 mg/L), EDTA (1 to 10 mM), ACD-A(0.01 to 10.0%) trehalose (0.01 to 10 gm/L), maltose (0.01 to 10.0 gm/L,dextran (0.01 to 3.0%, 100-500 MW), F-68 (0.001 to 10 mg/mL), sodium orpotassium sulfate (1 to 100 mM), sodium or potassium phosphate (1 to 100mM).

One suitable fixation process includes the following steps: Freezesubstitution prefix in −20° C. MeOH for 10 minutes; and postfixtreatment in EtOH, glyoxal, MeOH, and isopropanol. This process isbeneficial in that it avoids the use of formaldehyde and glutaraldehyde.Waste disposal issues are eliminated, and DNA and RNA retrieval is madeeasier because no cross linking occurs.

Genetic testing and fetal/maternal differentiation can now be performed.Standard FISH may be performed on the NRBCs that are found using theautomated cell identification algorithms. In addition, human TSIXsequences may be used to definitively identify the cells as fetal femaleand not maternal. TSIX expression stops on both human X chromosomebetween 2 and 4 years of age. Adult females do not express the TSIXgene. Thus, including a probe for TSIX in the FISH process will allow adefinitive determination of fetal female versus maternal status for allcandidate NRBCs being interrogated. A FISH signal at the TSIX region ofthe nucleus will only be present if the cell is fetal. The presence of aY chromosome determines if the nucleated red blood cell is from a malefetus.

It is also important to note that nuclei of the NRBCs are in the processof apoptosis and are being condensed in preparation for ejection fromthe cell. Highly condensed nuclei tend to have a lower efficiency fromFISH treatment than do non-condensed nuclei. Two novel approaches toimproving the FISH efficiency for condensed nuclei are:

(1) Perform FISH in a vacuum or under lower than atmospheric pressure;or

(2) Physically crush the cells and the nuclei prior to the applicationof the FISH probes by revisiting the XY locations of candidates andpressing on them in a controlled manner. The cells to be crushed orflattened would be the NRBCs identified by the DAPI/420 nm scan. On thesame microscope platform that did the DAPI/420 nm scan a motorizednosepiece could rotate over the “crushing head”. This could be, forexample, a spring loaded small diameter flat ended steel rod, andautomatically lowered onto the slide at the location of the NRBC to becrushed. The crushing force applied may be controlled by the springforce constant in the crushing rod. The rod diameter may be small, forexample, 100 microns in diameter so it would crush the target NRBC andthe perimeter of cells around it. An absolute XY location accuracy maynot be required. The small diameter also allows for very high crushingforces to be applied to the localized area.

The TSIX expression and the variation of TSIX expression versus humanage is described in Species Differences in TSIX/Tsix Reveal the Roles ofThese Genes in X-Chromosome Inactivation; Migeon, Barbara R.; Lee,Catherine H.; Chowdhury, Ashis K.; Carpenter, Heather;doi:10.1086/341605 (volume 71 issue 2 pp. 286-293).

Crushing cells to improve access to the nuclear material is described inCell Crushing: A Technique for Greatly Reducing Errors inMicrospectrometry; Davies, H. G; Wilkins, M. H. F; Boddy, R. G. H. B.;Experimental Cell Research 6/(550-553); 1954.

Novel aspects of the invention include: The combination of TSIX withFISH for the definitive determination of fetal female/maternal status ofcells; the use of vacuum to improve FISH results in fetal genetictesting; and the use of physical crushing of cells to improve access tothe nuclear material.

Stored XY locations may be used to record genetic test results. The XYlocations of the candidate NRBCs are used throughout the processing ofthe cells for genetic testing. It is possible to apply FISH probe toonly those cells of interest, thus reducing the overall cost per testper patient. It is also possible to revisit the cells and physicallyremove them from the substrate for physical segregation away from thepopulation of non-NRBCs, thus increasing the percentage of fetal DNArelative to maternal DNA.

It is possible to revisit the XY locations of the NRBC candidates tointerrogate the FISH results and fetal/maternal determination. It isalso possible to revisit the XY locations of the NRBC candidates andphysically decondense the nuclei.

The cells prepared by the methods described herein may be subject toantibody analysis. For example, several specific erythrocytic hemoglobinantibodies are available for the differential identification of fetalRBC's that occur in maternal peripheral blood (Zheng et al. 1999 Fetalcell identifiers: results of microscope slide-based immunocytochemicalstudies as a function of gestational age and abnormality. Am. J. Obstet.Gynecol. 180:1234-1239). Standard antibody staining techniques for fetaland embryonic hemoglobins may be performed on the NRBC's that are foundin maternal blood by filtering samples, preparing monolayers on slidesand locating NRBC's with the automated cell identification algorithmsoftware. Adults do not express the embryonic hemoglobin, epsilon, whilefetal RBC's may contain this embryonic hemoglobin up until the end ofthe first trimester (Mevron et al. 1999. Improved specificity of RBCdetection in chorionic villus sample supernatant fluids using anti-zetaand anti-epsilon monoclonal antibodies. Feta. Diagn. Ther. 14:291-295).Antibodies against other embryonic (zeta) and fetal hemoglobins may beused with anti-epsilon to increase the specificity of identification offetal NRBC's, but these antibodies will also recognize zeta and fetalhemoglobin expression in adult sickle cell anemics and thallesemics.Fetal hemoglobin is expressed during the last two trimesters ofpregnancy and shifts to beta-hemoglobin after birth.

EXAMPLE

FIG. 6 shows data obtained from maternal blood samples after enrichmentfor fetal nucleated cells according to one embodiment of the disclosure.The maternal blood samples were passed over leukocyte depletion filtersand cells remaining on the filter were eluted using elution buffer asdescribed above. The cells were smeared onto slides and stained withDAPI to detect nucleated cells and subject to immunohistochemistry usinganti epsilon hemoglobin antibody to detect the presence of fetal cells.A portion (% of sample analyzed) of the slides were illuminated with 420nm and UV light to distinguish nucleated from non-nucleated cells, andcells with hemoglobin from cells without hemoglobin. Cells wereclassified as non-nucleated red blood cells (RBC), white blood cells(WBC), and candidate nucleated red blood cells and the ratio of redblood cells to white blood cells (RBC:WBC) in the sample calculated. A %packing density was calculated based on the total number of cellscounted. Fetal nucleated Red Blood Cells in the fields analyzed wasconfirmed using anti epsilon hemoglobin antibody (#fnRBCs identified),and the predicted number of fetal nucleated red blood cells (#fnRBCsextrapolated) in each sample extrapolated.

As for additional details pertinent to the present invention, materialsand manufacturing techniques may be employed as within the level ofthose with skill in the relevant art. The same may hold true withrespect to method-based aspects of the invention in terms of additionalacts commonly or logically employed. Also, it is contemplated that anyoptional feature of the inventive variations described may be set forthand claimed independently, or in combination with any one or more of thefeatures described herein. Likewise, reference to a singular item,includes the possibility that there are plural of the same itemspresent. More specifically, as used herein and in the appended claims,the singular forms “a,” “and,” “said,” and “the” include pluralreferents unless the context clearly dictates otherwise. It is furthernoted that the claims may be drafted to exclude any optional element. Assuch, this statement is intended to serve as antecedent basis for use ofsuch exclusive terminology as “solely,” “only” and the like inconnection with the recitation of claim elements, or use of a “negative”limitation. Unless defined otherwise herein, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. The breadth of the present invention is not to be limited bythe subject specification, but rather only by the plain meaning of theclaim terms employed.

What is claimed is:
 1. A method of enriching for fetal nucleated cellsfrom a maternal blood sample, the method comprising: passing a maternalblood sample containing maternal nucleated cells and fetal nucleatedcells through a leukocyte depletion filter; retaining nucleated cellsincluding leukocytes on the filter; and eluting nucleated cells from thefilter with an elution buffer wherein the nucleated cells include fetalnucleated cells.
 2. The method of claim 1 further comprising stainingthe blood sample after passing through the leukocyte depletion filter.3. The method of claim 2 wherein staining comprises staining the bloodsample with a nuclear stain.
 4. The method of claim 1 further comprisingconcentrating and resuspending the blood sample before the passing step.5. The method of claim 4 wherein concentrating comprises concentratingby centrifugation.
 6. The method of claim 1 further comprising measuringthe blood sample after the eluting step.
 7. The method of claim 6further comprising standardizing an amount of cells in the blood sample.8. The method of claim 1 wherein the fetal nucleated cells comprisenucleated red blood cells.